Protocol 36494: Probe Assay - Homer2<em1(IMPC)J>
Version 1.0

Notes

Taqman qPCR protocols are run on a real time PCR instrument. Use an appropriate instrument specific Fluorophore/Quencher combination.

 

The genotyping protocol(s) presented here have been optimized for reagents and conditions used by The Jackson Laboratory (JAX). To genotype animals, JAX recommends researchers validate the assay independently upon receipt of animals into their facility. Reaction cycling temperature and times may require additional optimization based on the specific genotyping reagents used.

Expected Results

  

>chr7:81618675-81618777 103bp GCCTAACTTGCTGCCAATGT CTGGATGCCTGAGTGGAAGA

Mut = 99 bp

Wt = 103 bp

Fam = Mut

Hex = Wt

Sequence

Wt Sequence (deletions in lower case):

 GATATTCATAAGAACGCTTAGCAGCATTGACCTAGCAAACACCCCTATCTGGTTCCTGTGTTTTTAATAAGTGTGCTCATCTAACTGTCCGTAGTCCCGGGATCTGTCCATCATTCCTGTTTCTGACACTCTTTCAAGATCCTCAAGGACACTGAACTCCTTAGTCTCTGTTGTTTATAATTGTGACTTCTGTCCTGGGGTATTTAAGGAGATATTCAAGTTTGATAGGGATTGAACATAGAAAATAGGAAGGTAAAGTTTGAATTAAAAAGCTCTGAGTGGTGGCCCCAGgtatcatctggtcagcaaatcacaacttaccaatgctgtgtggagtgttctgggcccttaggccctgtgactgattgtcttggtcagcctttctgctggttagtctacactgcactctggttcaatgagtgggtttgtccagactcataaactcatattttctcattatgcctaacttgctgccaatgtaaggacaagcagtaacatgtgaagataattcactttggccaggaatctgggtgtgaaaccccgtcttccactcaggcatccagcgtcaatggcacagacgacgaaaaggcctctcacgcgagcccagccgacactcacctcaagtctgagaatgacaagctgaagatcgcgctgacacagaggtgaggttgcttcagcacacgcgcgcgcgcgcacacacacacacacacacacacacacacacacacacgcacgcgcacacacacacacacacacacaccccgctccctgcgctgtgtttcctgaaagcctttcccaccgggcagggtcctgctcagagcatcacattctgcctcctggtcagaggacgagcagttggcttcatcacctgtctgtcggtgtcagtgagcgtgtatgagcctcttggtgcagGGTGCGTGTACGGGAGATATAAATGTGAGTGTGTGTGTGAGAACCTGCTGGATCTCTAATTGTGTTTTGCTTTTGAAAGAGGAAGACCTACTTATTTATCTCTGACAAGAGAGAGTCTTAAACATCTAAGAAAAGTATGTAAGAGCCCAAGAAACAGTGCTTCTCTCTGAGGAGCCCACACCCACACTCTCAGGTGTGCCCTGTACCTTTCTATTAACCACTGTGTTTCAATAAACTCCAACTCTACTTTTAAAAAAAGTCTAAGAAATTATGAAAAATAA

This mutation is a 621 bp deletion beginning at Chromosome 7 position 81,618,326 bp and ending after 81,618,946 bp (GRCm38/mm10).

JAX Protocol

Protocol Primers

Primer 5' Label Sequence 5' → 3' 3' Label Primer Type Reaction Note
48894 GCC TAA CTT GCT GCC AAT GT Wild type Forward A
48895 CTG GAT GCC TGA GTG GAA GA Wild type Reverse A
48896 GAA TTA AAA AGC TCT GAG TGG TG Mutant Forward A
48897 CAA AAC ACA ATT AGA GAT CCA GCA G Mutant Reverse A
48898 Fluorophore-1 CCA GGA ATC TGG GTG TGA AA Quencher-1 WT Probe
48899 Fluorophore-2 CCC CAG GGT GCG TGT A Quencher-2 MUT Probe

Reaction A

Component Final Concentration
Kapa Probe Fast QPCR 1.00 X
ddH2O
48894 0.40 uM
48895 0.40 uM
48896 0.40 uM
48897 0.40 uM
Wt Probe 0.15 uM
Mutant Probe 0.15 uM
DNA

Cycling

Step Temp °C Time Note
1 95.0 --
2 95.0 --
3 60.0 --
4 -- repeat steps 2-3 for 40 cycles
5 40.0 -- Forever
JAX uses a very high speed Taq (~1000 bp/sec), use cycling times recommended for your reagents.

Strains Using This Protocol

This is the only strain that uses this protocol.